Autosomal dominant polycystic kidney disease (ADPKD) is a channelopathy affecting multiple organs and tissues. Mutations in two separate, but genetically interacting loci, pkd1 and pkd2, are responsible for the vast majority of all cases of ADPKD. Polycystin-2 (PC2) or PKD2 belongs to the transient receptor potential (TRP) superfamily of ion channels. Homozygous deletion of pkd2 in mice results in embryonic death due to kidney and heart defects representing perhaps one of the most severe phenotypes of all known TRP channels to date. However, little is known about the mechanisms underlying fundamental properties such as molecular assembly, gating, and modes of activation. We have shown that PKD2 physically interacts with other channel subunits such as TRPC1, and auxiliary proteins such as PKD1 and the mammalian homolog of diaphanous-related formin, mdia1. Our functional data show that PKD2 forms an EGF- activated plasma membrane channel in kidney epithelial cells. Mechanistically, EGF activates PKD2 in a voltage-dependent manner and through the action of phospholipase C (PLC)-gamma2 and phosphoinositide 3-kinase (PI3K). New preliminary data indicate that mdia1 functions as a voltage-dependent gate for PKD2 by specifically inhibiting its activity at negative (hyperpolarizing) but not positive (depolarizing) potentials. The voltage dependent action of mdia1 is caused by the molecular switching from its autoinhibited state at negative potentials to its activated state at positive potentials. We therefore hypothesize that PKD2 forms a receptor- operated channel complex whose activity is dependent on protein-protein interactions with other channels (TRPC1) and auxiliary subunits (PKD1, mdia1, and PLC-gamma2). The specific aims of our proposal are to determine the mechanism(s) by which (1) PLC-gamma2 and its substrate phosphatidylinositol-4,5- bisphosphate (PIP2), (2) mdia1, and (3) TRPC1 regulate PKD2 channel activity. These aims will be mainly accomplished by the biochemical identification of the interacting domains in PKD2 with PLC-gamma2, PIP2, mdia1, and TRPC1 and their functional characterization by electrophysiology. It is almost certain that perturbation of PKD2 channel activity per se is one of the major causes of ADPKD pathophysiology. The proposed studies will help us understand PKD2 channel function and regulation and provide insights into the mechanisms by which naturally occurring mutations alter its activity.